Human deficiency virus type-1 (HIV-1) peptide encoded by the VIF gene

ABSTRACT

This invention is in the field of lymphadenopathy virus. This invention relates to a diagnostic means and method to detect the presence of DNA, RNA or antibodies of the lymphadenopathy retrovirus associated with the acquired immune deficiency syndrome or of the lymphadenopathy syndrome by the use of DNA fragments or the peptides encoded by said DNA fragments. The invention further relates to the DNA fragments, vectors comprising them and the proteins expressed.

This is a division of application Ser. No. 09/344,449 filed Dec. 31,1997, now U.S. Pat. No. 6,261,564 which is a division of applicationSer. No. 08/195,024, filed Feb. 14, 1994, now U.S. Pat. No. 5,705,612,which is a division of application Ser. No. 07/953,060, filed Nov. 5,1992, now abandoned, which is a division of application Ser. No.07/158,652, filed Feb. 22, 1988, which is a division of application Ser.No. 06/771,248, filed Aug. 30, 1985, now abandoned, all of which areincorporated herein by reference.

The invention relates to cloned DNA sequences indistinguishable fromgenomic RNA and DNA of lymphadenopathy-associated virus (LAV), a processfor their preparation and their uses. It relates more particularly tostable probes including a DNA sequence which can be used for thedetection of the LAV virus or related viruses or DNA proviruses in anymedium, particularly biological samples containing any of them. Theinvention also relates to polypeptides, whether glycosylated or not,encoded by said DNA sequences.

Lymphadenopathy-associated virus (LAV) is a human retrovirus firs'tisolated from the lymph node of a homosexual patient withlymphadenopathy syndrome, frequently a prodrome or, a benign form ofacquired immune deficiency syndrome (AIDS). Subsequently other LAVisolates have been recovered from patients with AIDS or pre-AIDS. Allavailable data are consistent with the virus being the causative agentof AIDS.

A method for cloning such DNA sequences has already been disclosed inBritish Patent Application Nr. 84 23659 filed on Sep. 19, 1984.Reference is here-after made to that application as concerns subjectmatter in common with the further improvements to the inventiondisclosed herein.

The present invention aims at providing additional new means whichshould not only also be useful for the detection of LAV or relatedviruses (hereafter more generally referred to as “LAV viruses”), butalso have more versatility, particularly in detecting specific parts ofthe genomic DNA of said viruses whose expression products are not alwaysdirectly detectable by immunological methods.

The present invention further aims at providing polypeptides containingsequences in common with polypeptides encoded by the LAV genomic RNA. Itrelates even more particularly to polypeptides comprising antigenicdeterminants included in the proteins encoded and expressed by the LAVgenome occuring in nature. An additional object of the invention is tofurther provide means for the detection of proteins related to LAVvirus, particularly for the diagnosis of AIDS or pre-AIDS or, to thecontrary, for the detection of antibodies against the LAV virus orproteins related therewith, particularly in patients afflicted with AIDSor pre-AIDS or more generally in asymtomatic carriers and inblood-related products. Finally the invention also aims at providingimmunogenic polypeptides, and more particularly protective polypeptidesfor use in the preparation of vaccine compositions against AIDS orrelated syndroms.

The present invention relates to additional DNA fragments, hybridizablewith the genomic RNA of LAV as they will be disclosed hereafter, as wellas with additional cDNA variants corresponding to the whole genomes ofLAV viruses. It further relates to DNA recombinants containing said DNAsor cDNA fragments.

The invention relates more particularly to a cDNA variant correspondingto the whole of LAV retroviral genomes, which is characterized by aseries of restriction sites in the order hereafter (from the 5′ end tothe 3′ end).

The coordinates of the successive sites of the whole LAV genome(restriction map) are indicated hereafter too, with respect to the HindIII site (selected as of coordinate 1) which is located in the R region.The coordinates are estimated with an accuracy of ±200 bp:

Hind III 0 Sac I 50 Hind III 520 Pst I 800 Hind III 1 100 Bgl II 1 500Kpn I 3 500 Kpn I 3 900 Eco RI 4 100 Eco RI 5 300 Sal I 5 500 Kpn I 6100 Bgl II 6 500 Bgl II 7 600 Hind III 7 850 Bam HI 8 150 Xho I 8 600Kpn I 8 700 Bgl II 8 750 Bgl II 9 150 Sac I 9 200 Hind III 9 250

Another DNA variant according to this invention optionally contains anadditional Hind III approximately at the 5 550 coordinate.

Reference is further made to FIG. 1 which shows a more detailedrestriction map of said whole-DNA (λJ19).

An even more detailed nucleotidie sequence of a preferred DNA accordingto the invention is shown in FIGS. 4-12 hereafter.

The invention further relates to other preferred DNA fragments whichwill be referred to hereafter.

Additional features of the invention will appear in the course of thenon-limitative disclosure of additional features of preferred DNAs ofthe invention, as well as of preferred polypeptides according to theinvention. Reference will further be had to the drawings in which:

FIG. 1 is the restriction map of a complete LAV genome (clone λJ19);

FIGS. 2 and 3 show diagrammatically parts of the three possible readingphases of LAV genomic RNA, including the open reading frames (ORF)apparent in each of said reading phases

FIGS. 4-12 show the successive nucleotidic sequences of a complete LAVgenome. The possible peptidie sequences in relation to the threepossible reading phases related to the nucleotidie sequences shown arealso indicated

FIGS. 13-18 reiterate the sequence of part of the LAV genome containingthe genes coding for the enveloppe proteins, with particular boxedpeptidic sequences which corresponds to groups which normally carryglycosyl groups.

The sequencing and determination of sites of particular interest wascarried out on a phage recombinant corresponding to λJ19 disclosed inthe abovesaid British Patent application Nr. 84 23659. A method forpreparing it is disclosed in that application.

The whole recombinant phage DNA of clone λJ19 (disclosed in the earlierapplication) was sonicated according to the protocol of DEININGER(1983). Analytical Biochem. 129, 216, the DNA was repaired by a Klenowreaction for 12 hours at 16° C. The DNA was electrophoresed through 0.8%agarose {overscore (g)}el and DNA in the size range of 300-600 bp wascut out and electroeluted and precipitated. Resuspended DNA (in 10 mMTris, pH 8; 0,1 mM EDTA) was ligated into M13mp8 RF DNA (cut by therestriction enzyme SmaI and subsequently alkaline phosphated), using T4DNA- and RNA-ligases (Haniatis T et al (1982)—Molecular cloning—ColdSpring Harbor Laboratory). An E. coli strain designated as TG1 was usedfor further study. This strain has the following genotype: Δlac pro,supE, thi.F′traD36, proAB, lacI^(q), ZΔM15,r⁻

This E. coli TGI strain has the peculiarity of enabling recombinants tobe recognized easily. The blue colour of the cells transfected withplasmids which did not recombine with a fragment of LAV DNA is notmodified. To the contrary cells transfected by a recombinant plasmidcontaining a LAV DNA fragment yield white colonies. The technique whichwas used is disclosed in Gene (1983), 26. 101.

This strain was transformed with the ligation mix using the Hanahanmethod (Hanahan D (1983) J. Mol. Biol. 166, 557). Cells were plated outon tryptone-agarose plate with IPTG and X-gal in soft agarose. Whiteplaques were either picked and screened or screened directly in situusing nitrocellulose filters. Their DNAs were hybridized withnick-translated DNA inserts of pUC18 Hind III subclones of λJ19, thispermitted the isolation of the plasmids or subclones of λ which areidentified in the table hereafter. In relation to this table it shouldalso be noted that the designation of each plasmid is followed by thedeposition number of a cell culture of E. coli TGI containing thecorresponding plasmid at the “Collection Nationale des Cultures deMicro-organismes” (C.N.C.M.) of the Pasteur Institute in Paris, France.A non-transformed TGI cell line was also deposited at the C.N.C.M. underNr. I-364. All these deposits took place on Nov. 15, 1984. The sizes ofthe corresponding inserts derived from the LAV genome have also beenindicated.

TABLE Essential features of the recombinant plasmids pJ19 - 1 plasmid(I-365) 0.5 kb Hind III - Sac I - Hind III pJ19 - 17 plasmid (I-367) 0.6kb Hind III - Pst 1 - Hind III pJ19 - 6 plasmid (I-366) 1.5 kb Hind III(5′) Bam HI Xho I Kpn I Bgl II Sac I (3′) Hind III pJ19 - 13 plasmid(I-368) 6.7 kb Hind III (5′) Bgl II Kpn I Kpn I Eco RI Eco RI Sal I KpnI Bgl II Bgl II Hind III (3′)

Positively hybridizing M13 phage plates were grown up for 5 hours andthe single-stranded DNAs were extracted.

M13mp8 subcldnes of λJ19 DNAs were sequenced according to the dideoxymethod and technology devised by Sanger et al (Sanger et al (1977),Proc. Natl. Acad. Sci. USA, 74, 5463 and M13 cloning and sequencinghandbook, AMERSHAM (1983), the 17-mer oligonucleotide primer α-³⁵SdATP(400 Ci/mmol, AMERSHAM), and 0.5X-5X buffer gradient gels (Biggen M.D.et al (1983, Proc. Natl. Acad. Sci. USA, 50, 3963) were used. Gels wereread and put into the computer under the programs of Staden (Staden R.(1982), Nucl. Acids Res. 10, 4731). All the appropriate references andmethods can be found in the AMERSHAM M13 cloning and sequencinghandbook.

The complete sequence of λJ19 was deduced from the experiments asfurther disclosed hereafter.

FIGS. 4-12 provide the DNA nucleotidie sequence of the complete genomeof LAV. The numbering of the nucleotides starts from a left most HindIII restriction site (5′AAG . . . ) of the restriction map. Thenumbering occurs in tens whereby the last zero number of each of thenumbers occuring on the drawings is located just below the nucleotidecorresponding to the nucleotides designated. I.e. the nucleotide atposition 10 is T, the nucleotide at position 20 is C, etc.

Above each of the lines of the successive nucleotidic sequences thereare provided three lines of single letters corresponding to theaminoacid sequence deduced from the DNA sequence (using the geneticcode) for each at the three reading phases, whereby said single lettershave the following meanings.

A: alanine

R: arginine

K: lysine

H: histidine

C: cysteine

M: methionine

W: tryptophan

F: phenylalanine

Y: tyrosine

L: leucine

V: valine

I: isoleucine

G: glycine

T: threonine

S: serine

E: glutamic acid

D: Aspartic acid

N: asparagine

Q: glutamine

P: proline.

The asterik signs “*” correspond to stop codons (i.e. TAA, TAG and TGA).

Starting above the first line of the DNA nucleotidic sequence of FIG. 4the three reading phases are respectively marked “1”, “2”, “3”, on theleft handside of the drawing. The same relative presentation of thethree theoritical reading phases is then used all over the successiveslines of the LAV nucleotidic sequence.

FIGS. 2 and 3 provide a diagrammatized representation of the lengths ofthe successive open reading frames corresponding to the successivereading phases (also referred to by numbers “1”, “2” and “3” appearingin the left handside part of FIG. 2). The relative positions of theseopen reading frames (ORF) with respect to the nucleotidic structure ofthe LAV genome is referred to by the scale of numbers representative ofthe respective positions of the corresponding nucleotides in the DNAsequence. The vertical bars correspond to the positions of thecorresponding stop codons.

1) The “Gag Gene” (or ORF-gaq)

The “gag gene” codes for core proteins. Particularly it appears that agenomic fragment (ORF-gag) thought to code for the core antigensincluding the p25, p18 and p13 proteins is located between nucleotidicposition 236 (starting with 5′ CTA GCG GAG 3′) and nucleotidic position1759 (ending by CTCG TCA CAA 3′). The structure of the peptides orproteins encoded by parts of said ORF is deemed to be that correspondingto phase 2.

The methionine aminoacid “M” coded by the ATG at position 260-262 is theprobable initiation methionine of the gag protein precursor. The end ofORF-gag and accordingly of gag protein appears to be located at position1759.

The beginning of p25 protein, thought to start by aP-I-V-Q-N-I-Q-G-Q-M-V-H . . . aminoacid sequence is thought to be codedfor by the nucleotidic sequence CCTATA . . . starting at position 656.

Hydrophilic peptides in the gag open reading frame are identifiedhereafter. They are defined starting from aminoacid 1=Met (M) coded bythe ATG starting from 260-2 in the LAV DNA sequence.

Those hydrophilic peptides are

12-32 aminoacids inclusive

37-46 aminoacids inclusive

49-79 aminoacids inclusive

88-153 aminoacids inclusive

158-165 aminoacids inclusive

178-188 aminoacids inclusive

200-220 aminoacids inclusive

226-234 aminoacids inclusive

239-264 aminoacids inclusive

288-331 aminoacids inclusive

352-361 aminoacids inclusive

377-390 aminoacids inclusive

399-432 aminoacids inclusive

437-484 aminoacids inclusive

492-498 aminoacids inclusive

The invention also relates to any combination of these peptides.

2) The “Pol Gene” (or ORF-pol)

FIGS. 4-12 also show that the DNA fragments extending from nucleotidicposition 1555 (starting with 5′TTT TTT . . . 3′ to nucleotidic position5086 is thought to correspond to the pol gene. The polypeptidicstructure of the corresponding polypeptides is deemed to be thatcorresponding to phase 1. It stops at position 4563 (end by 5′G GAT GAGGAT 3′).

These genes are thought to code for the virus polymerase or reversetranscriptase.

3) The Envelope Gene (or ORF-env)

The DNA sequence thought to code for envelope proteins is thought toextend from nucleotidic position 5670 (starting with 5′AAA GAG GAG A . .. 3′) up to nucleotidic position 8132 (ending by . . . A ACT AAA GAA3′). Polypeptidic structures of sequences of the envelope proteincorrespond to those read according to the “phase 3” reading phase.

The start of env transcription is thought to be at the level of th ATGcodon at positions 5691-5693.

Additional feature of the envelope protein coded by the env genes appearon FIGS. 13-18. These are to be considered as paired FIGS. 13 and 14; 15and 16; 17 and 18 respectively.

It is to be mentioned that because of format difficulties.

FIG. 14 overlaps to some extent with FIG. 13.

FIG. 16 overlaps to some extent with FIG. 15.

FIG. 18 overlaps to some extent with FIG. 17.

Thus for instance FIGS. 13 and 14 must be considered together.Particularly the sequence shown on the first line on the top of FIG. 13overlaps with the sequence shown on the first line on the top of FIG.14. In other words the starting of the reading of the successivesequences of the env gene as represented in FIGS. 13-18 involves firstreading the first line at the top of FIG. 13 then proceeding furtherwith the first line of FIG. 14. One then returns to the beginning of thesecond line of FIG. 13, then again further proceed with the reading ofthe second line of page 14, etc. . . . The same observations then applyto the reading of the paired FIGS. 15 and 16, and paired FIGS. 17 and18, respectively.

The locations of neutralizing epitopes are further apparent in FIGS.13-18, reference is more particularly made to the boxed groups of threeletters included in the aminoacid sequences of the envelope proteins(reading phase 3) which can be designated generally by the formula N-X-Sor N-X-T, wherein X is any other possible aminoacid. Thus the initialprotein product of the env gene in a glycoprotein of molecular weight inexcess of 91,000. These groups are deemed to generally carryglycosylated groups. These N-X-S and N-X-T groups with attachedglycosylated groups form together hydrophylic regions of the protein andare deemed to be located at the periphery of and to be exposed outwardlywith respect to the normal conformation of the proteins. Consequentlythey are considered as being epitopes which can efficiently be broughtinto play in vaccine compositions.

The invention thus concerns with more particularity peptide sequencesincluded in the env-proteins and excizable therefrom (or having the sameaminoacid structure), having sizes not exceeding 200 aminoacids.

Preferred peptides of this invention (referred to hereafter as a, b, c,d, e, f) are deemed to correspond to those encoded by the nucleotidesequences which extend respectively between the following positions:

a) from about 6095 to about 6200

b) from about 6260 to about 6310

c) from about 6390 to about 6440

d) from about 6485 to about 6620

e) from about 6860 to about 6930

f) from about 7535 to about 7630

Other hydrophilic peptides in the env open reading frame are identifiedhereafter, they are defined starting from aminoacid 1=lysine (K) codedby the AAA at position 5670-2 in the LAV DNA sequence.

These hydrophilic peptides are

8-23 aminoacids inclusive

63-78 aminoacids inclusive

82-90 aminoacids inclusive

97-123 aminoacids inclusive

197-201 aminoacids inclusive

239-294 aminoacids inclusive

300-327 aminoacids inclusive

334-381 aminoacids inclusive

397-424 aminoacids inclusive

466-500 aminoacids inclusive

510-523 aminoacids inclusive

551-577 aminoacids inclusive

621-630 aminoacids inclusive

657-679 aminoacids inclusive

719-758 aminoacids inclusive

780-803 aminoacids inclusive

The invention also relates to any combination of these peptides.

4) The Other ORF

The invention further concerns DNA sequences which provide open readingframes defined as ORF-Q, ORF-R and as “1”, “2”, “3”, “4”, “5”, therelative position of which appears more particularly in FIGS. 2 and 3.

These ORFs have the following locations:

ORF-Q phase 1 start 4478 stop 5086 ORF-R phase 2 start 8249 stop 8896ORF-1 phase 1 start 5029 stop 5316 ORF-2 phase 2 start 5273 stop 5515ORF-3 phase 1 start 5383 stop 5616 ORF-4 phase 2 start 5519 stop 5773ORF-5 phase 1 start 7966 stop 8279

The LTR (long terminal repeats) can be defined as lying between position8560 and position 160 (end extending over position 9097/1). As a matterof fact the end of the genome is at 9097 and, because of the LTRstructure of the retrovirus, links up with the beginning of thesequence:

The invention concerns more particularly all the DNA fragments whichhave been more specifically referred to hereabove and which correspondto open reading frames. It will be understood that the man skilled inthe art will be able to obtain them all, for instance by cleaving anentire DNA corresponding to the complete genome of a LAV species, suchas by cleavage by a partial or complete digestion thereof with asuitable restriction enzyme and by the subsequent recovery of therelevant fragments. The different DNAs disclosed in the earliermentioned British Application can be resorted to also as a source ofsuitable fragments. The techniques disclosed hereabove for the isolationof the fragments which were then included in the plasmids referred tohereabove and which were then used for the DNA sequencing can be used.

Of course other methods can be used. Some of them have been examplifiedin the earlier British Application. reference is for instance made tothe following methods.

a) DNA can be transfected into mammalian cells with appropriateselection markers by a variety of techniques, calcium phosphateprecipitation, polyethylene glycol, protoplast-fusion, etc.

b) DNA fragments corresponding to genes can be cloned into expressionvectors for E. coli, yeast- or mammalian cells and the resultantproteins purified.

c) The provival DNA can be “shot-gunned” (fragmented) into procaryoticexpression vectors to generate fusion polypeptides. Recombinantproducing antigenically competent fusion proteins can be identified bysimply screening the recombinants with antibodies against LAV antigens.

The invention also relates more specifically to cloned probes which canbe made starting from any DNA fragment according to this invention, thusto recombinant DNAs containing such fragments, particularly any plasmidsamplifiable in procaryotic or eucaryotic cells and carrying saidfragments.

Using the cloned DNA fragments as a molecular hybridization probe—eitherby marking with radionucleotides or with fluorescent reagents—LAV virionRNA may be detected directly in the blood, body fluids and bloodproducts (e.g. of the antihemophylic factors such as Factor VIIIconcentrates) and vaccines, i.e. hepatitis 8 vaccine. It has alreadybeen shown that whole virus can be detected in culture supernatants ofLAV producing cells. A suitable method for achieving that detectioncomprises immobilizing virus onto said a support e.g. nitrocellulosefilters, etc., disrupting the virion and hybridizing with labelled(radiolabelled or “cold” fluorescent- or enzyme-labelled) probes. Suchan approach has already been developed for Hepatitis B virus inperipheral blood (according to SCOTTO J. et al. Hepatology (1983), 3,379-384).

Probes according to the invention can also be used for rapid screeningof genomic DNA derived from the tissue of patients with LAV relatedsymptoms, to see if the proviral DNA or RNA is present in host tissueand other tissues.

A method which can be used for such screening comprise the followingsteps: extraction of DNA from tissue, restriction enzyme cleavage ofsaid DNA, electrophoresis of the fragments and Southern blotting ofgenomic DNA from tissues, subsequent hybridization with labelled clonedLAV provival DNA. Hybridization in situ can also be used.

Lymphatic fluids and tissues and other non-lymphatic tissues of humans,primates and other mammalian species can also be screened to see ifother evolutionnary related retrovirus exist. The methods referred tohereabove can be used, although hybridization and washings would be doneunder non stringent conditions.

The DNA according to the invention can be used also for achieving theexpression of LAV viral antigens for diagnostic purposes.

The invention also relates to the polypeptides themselves which can beexpressed by the different DNAs of the inventions, particularly by theORFs or fragments thereof, in appropriate hosts, particularlyprocaryotic or eucaryotic hosts, after transformation thereof with asuitable vector previously modified by the corresponding DNAs.

These polypeptides can be used as diagnostic tools, particularly for thedetection of antibodies in biological media, particularly in sera ortissues of persons afflicted with pre-AIDS or AIDS, or simply carryingantibodies in the absence of any apparent disorders. Conversely thedifferent peptides according to this invention can be used themselvesfor the production of antibodies, preferably monoclonal antibodiesspecific of the different peptides respectively. For the production ofhybridomas secreting said monoclonal antibodies conventional productionand screening methods are used. These monoclonal antibodies, whichthemselves are part of the invention then provide very useful tools forthe identification and even determination of relative proportions of thedifferent polypeptides or proteins in biological samples, particularlyhuman samples containing LAV or related viruses.

Thus all of the above peptides can be used in diagnostics as sources ofimmunogens or antigens free of viral particles, produced usingnon-permissive systems, and thus of little or no biohazard risk.

The invention further relates to the hosts (procaryotic or eucaryoticcells) which are transformed by the above mentioned recombinants andwhich are capable of expressing said DNA fragments.

Finally it also relates to vaccine compositions whose active principleis to be constituted by any of the expressed antigens, i.e. wholeantigens, fusion polypeptides or oligopeptides in association with asuitable pharmaceutical or physiologically acceptable carrier.

Preferably the active principles to be considered in that field consistof the peptides containing less than 250 aminoacid units, preferablyless than 150 as deducible for the complete genomas of LAV, and evenmore preferably those peptides which contain one or more groups selectedfrom N-X-S and N-X-T as defined above. Preferred peptides for use in theproduction of vaccinating principles are peptides (a) to (f) as definedabove. By way of example having no limitative character, there may bementioned that suitable dosages of the vaccine compositions are thosewhich enable administration to the host, particularly human host rangingfrom 10 to 500 micrograms per kg, for instance 50 to 100 micrograms perkg.

For the purpose of clarity FIGS. 19 to 26 are added, reference may bemade thereto in case of difficulties of reading blurred parts of FIGS. 4to 12.

Needless to say that FIGS. 19-26 are merely a reiteration of the wholeDNA sequence of the LAV genoma.

Finally the invention also concerns vectors for the transformation ofeucaryotic cells of human origin, particularly lymphocytes, thepolymerases of which are capable of recognizing the LTRs of LAV.Particularly said vectors are characterized by the presence of a LAV LTRtherein, said LTR being then active as a promoter enabling the efficienttranscription and translation in a suitable host of the above defined,of a DNA insert coding for a determined protein placed under itscontrols.

Needless to say that the invention extends to all variants of genomesand corresponding DNA fragments (ORFs) having substantially equivalentproperties, all of said genomes belonging to retroviruses which canbeconsidered as equivalents of LAV.

What is claimed is:
 1. A purified peptide encoded by the vpr gene ofHIV-1, wherein the peptide is free of particles of said virus and hasthe following amino acid sequence:Met-Glu-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Asp-Pro-His-Asn-Glu-Trp-Thr-Leu-Gln-Leu-Leu-Glu-Glu-Leu-Lys-Asn-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Gly-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-Leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Gln-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser.2. A peptide of HIV-1 expressed from DNA corresponding to the vpr openreading frame, the DNA having the following nucleotide sequence:5030       5040       5050       5060       5070   AT GGAACAAGCCCCAGAAGACC AAGGGCCACA GAGGGAGCCA      5080       5090       5100       5110       5120 CACAAGTAAGGGACACTAGA GCTTTTAGAG GAGCTTAAGA ATGAAGCTGT      5130       5140       5150       5160       5170 TAGACATTTTCCTAGGATTT GGCTCCATGG CTTAGGGCAA CATATCTATG      5180       5190       5200       5210       5220 AAACTTATGGGGATACTTGG GCAGGAGTGG AAGCCATAAT AAGAATTCTG      5230       5240       5250       5260       5270 CAACAACTGCTGTTTATCCA TTTCAGAATT GGGTGTCGAC ATAGCAGAAT      5280       5290       5300       5310 AGGCGTTACT CAACAGAGGAGAGCAAGAAA TGGAGCCAGT AGATCC,

wherein the DNA is a recombinant DNA molecule.